Despite dramatic treatment advances in the past few decades, there remain subsets of pediatric leukemia that are very difficult to treat. A striking example is a particularly aggressive type of acute myeloid leukemia (AML) caused by rearrangement of the genes NUP98 (nucleoporin, 98-kd component of nuclear pore complex) and NSD1 (nuclear receptor-binding SET domain protein 1). This rearrangement leads to fusion of the normally separate NUP98 and NSD1 proteins. The first NUP98-NSD1 fusion was identified less than fifteen years ago, and it has since become clear that these genetic lesions are often missed by routine genetic testing of AML patients. Recent comprehensive studies using a specific detection method found that AML harboring NUP98- NSD1 was present in 4-5% of pediatric AML, associated with a grim 4-year event-free survival rate of 10-30%. Thus novel therapies specifically targeted to this group of pediatric patients are urgently needed. AML harboring NUP98-NSD1 fusions is initiated through activation of the expression of specific cancer-causing oncogenes, which results in a failure of the normal process of cell differentiation to give specific types of blood cell. Undifferentiated cells acquire the ability to proliferate unchecked, and it is this acquired capacity for self- renewal that is the key trigger for these cases of pediatric leukemia. NSD1 is a methyltransferase enzyme that activates gene expression by methylating a specific lysine residue in histones, affecting their interaction with DNA. Therefore, small molecules that inhibit the enzyme activity of NSD1 should be effective in reversing activation of the specific genes causing AML and allow malignant cells to revert to a normal differentiated form. Our goal is to develop a suite of assays to enable high-throughput screening of several hundred thousand compounds to discover inhibitors of the NSD1 enzyme, and to characterize these inhibitors to identify selective chemical probes and lead candidates as molecular therapies targeted to childhood leukemia's harboring the NUP98-NSD1 fusion.